New Pyrazolopyrimidine Inhibitors of Protein Kinase D as Potent Anticancer Agents for Prostate Cancer Cells
Figure 8
1-NA-PP1 blocked prostate cancer cell migration and invasion.
A.1-NA- PP1 blocked prostate cancer cell migration. PC3 cells were grown to confluence in 6-well plates. Monolayer was wounded and imaged immediately (0 h). Cells were then treated in growth media containing a vehicle (DMSO) or 30 µM of 1-NA-PP1 for 22 h and wound closure was measured. Percentage wound healing was calculated as the percent of healed wound area as compared to the original wound. B. 1-NA-PP1 inhibited prostate cancer cell invasion. DU145 cells were incubated with 30 µM 1-NA-PP1 in Matrigel inserts. After 20 h, noninvasive cells were removed and invasive cells were fixed in 100% methanol, stained in 0.4% hematoxylin solution, and photographed. The number of cells that invaded the Matrigel matrix was determined by cell counts in 6 fields relative to the number of cells that migrated through the control insert. Percentage invasion was calculated as the percent of the cells invaded through Matrigel inserts vs. the total cells migrated through the control inserts. C. Overexpressed PKD1 and PKD3 reversed the inhibitory effects of 1-NA-PP1 on tumor cell invasion. DU145 cells were infected with null, PKD1, and PKD3 adenoviruses (Adv-null, Adv-PKD1, and Adv-PKD3) at 100 MOI. After 24 h, cells were replated in control and Matrigel inserts, and a Matrigel invasion assay was conducted as described above. The overexpression of PKD1 and PKD3 was confirmed by Western blotting analysis (images below the graphs). All the above experiments were repeated at least three times and data from a representative experiment are shown.